Thermodynamic and structural characteristics of the Escherichia coli DnaT protein trimerization reaction have been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. Binding of magnesium to the DnaT monomers regulates the intrinsic affinity of the DnaT trimerization reaction. Comparison between the DnaT trimer and the isolated N-terminal core domain suggests that magnesium binds to the N-terminal domain but does not associate with the C-terminal region of the protein. The magnesium binding process is complex and involves approximately three Mg2+ cations per protein monomer. The observed effect seems to be specific for Mg2+. In the examined salt concentration range, monovalent cations and anions do not affect the trimer assembly process. However, magnesium affects neither the cooperativity of the trimerization reaction nor the GnHCl-induced trimer dissociation, strongly indicating that Mg2+ indirectly stabilizes the trimer through the induced changes in the monomer structures. Nevertheless, formation of the trimer also involves specific conformational changes of the monomers, which are independent of the presence of magnesium. Binding of Mg2+ cations dramatically changes the thermodynamic functions of the DnaT trimerization, transforming the reaction from a temperature-dependent to temperature-independent process. Highly cooperative dissociation of the trimer by GnHCl indicates that both interacting sites of the monomer, located on the N-terminal core domain and formed by the small C-terminal region, are intimately integrated with the entire protein structure. In the intact protein, the C-terminal region most probably interacts with the corresponding binding site on the N-terminal domain of the monomer. Functional implications of these findings are discussed.
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